This invention relates to the establishment of the first and therefore novel method of culturing sponge cells, coral cells and cells from other invertebrates in vitro. The cells cultured in vitro, which can be cultured as units similar to aggregates, are referred to as primmorphs. A method is thus available which makes it possible for the first time, using cells/aggregates/primmorphs from sponges, corals and other invertebrates, to introduce methods: (i) of preparing substances which modulate proliferation and DNA synthesis, (ii) of identifying/detecting environmentally harmful substances, (iii) of culturing bacteria and other micro-organisms, (iv) of preparing asexual reproductive bodies that can be used in aquaculture for growing corresponding organisms, (v) of preparing cell libraries, (vi) of optimising the nutritional requirements of the cells/aggregates/primmorphs, and (vii) of identifying substances which modulate telomerase activity in cells/aggregates/primmorphs.
The phylum Porifera (sponges), together with the other metazoan phyla has a monophyletic origin, [Mxc3xcller W. E. G. (1995) Naturwissenschaften 82, 321-329]. A fundamental autapomorphic feature of the metazoa, including the Porifera, is, for example, the presence of the receptor tyrosine kinase, which can only be found in metazoa [Mxc3xcller W. E. G., Schxc3xa4cke H. (1996) Prog. Molec. Subcell. Biol. 17, 183-208].
Within the metazoa, the Porifera exhibit a plesiomorphic feature which can be found in no other higher phylum of metazoa; all (or almost all) their cells have a high level of telomerase activity [Koziol C., Borojevic R., Steffen R., Mxc3xcller W. E. G. (1998) Mech. Ageing Develop. 100, 107-120]. In principle, this is an indication that sponge cells cannot be subdivided into gametes or somatic cells [Mxc3xcller W. E. G. (1998a) Progr. Molec. Subcell. Biol. 19, 98-132; Mxc3xcller W. E. G. (1998b) Naturwiss. 85:11-25]. In higher metazoa not suffering from tumours, virtually only the gametes are always telomerase-positive, whereas the somatic cells are telomerase-negative [Lange T. v. (1998) Science 279,334-335].
Because of this property that all (or virtually all) sponge cells are telomerase-positive, it might be presumed that sponge cells are immortal. So far, however, there has not yet been any report of neoplastic diseases in sponges [De-Flora S., Bagnasco M., Bennicelli C., Camoirano A., Bojnemirski A., Kurelec B. (1995). Mutagenesis 10, 357-364]. In the very first report on the high telomerase activity in sponges, it was shown by our group that, when removed from their associated tissue and converted into a dissociated state, the cells become telomerase-negative [Koziol C., Borojevic R., Steffen R., Mxc3xcller W. E. G. (1998) Mech. Ageing Develop. 100, 107-120]. Cells in a single-cell suspension very probably die off through apoptosis [Wagner C., Steffen R., Koziol C., Batel R., Lacorn M., Steinhart H., Simat T., Mxc3xcller W. E. G. (1998) Marine Biol. 131, 411-421]. In addition, the fact that sponges have a species-specific blueprint led us to postulate that sponges are equipped with an apoptosis mechanism to replace a certain group of cells at a specific time. This assumption was supported by the finding that cells in the sponge tissue are induced to effect apoptosis in response to endogenous factors (e.g. the addition of heat-treated bacteria) and to exogenous factors (cadmium) [Wagner C., Steffen R., Koziol C., Batel R., Lacorn M., Steinhart H., Simat T., Mxc3xcller W. E. G. (1998) Marine Biol. 131,411-421].
After it had been demonstrated by our group that sponge cells possess high telomerase activity [Koziol C., Borojevic R., Steffen R., Mxc3xcller W. E. G. (1998) Mech. Ageing Develop. 100, 107-120], it appeared an easily achievable aim to establish a sponge cell culture. Up to now, however, it has only been possible to keep sponge cells alive, as in the case of the species Hymeniacidon heliophila [Pomponi S. A., Willoughby R. (1994) In: R. van Soest, A. A. Balkema (Eds.) Sponges in Time and Space, Rotterdam, Brookfield, pp.395-400], Latrunculia magnifica [Ilan M., Contini H., Carmeli S., Rinkevich B. (1996) J. Mar. Biotechnol. 4, 145-149] and Suberites domuncula [Mxc3xcller W. E. G., Steffen R., Rinkevich B., Matranga V., Kurelec B. (1996) Marine Biol. 125, 165-170]. These cells do not, however, proliferate [Ilan M., Contini H., Carmeli S., Rinkevich B. (1996) J. Mar. Biotechnol. 4, 145-149].
One reason why, when kept in vitro, sponge cells have so far only remained in a quiescent state is, for example, that the methods of establishing the single-cell culture have been inappropriate, i.e. suitable culture conditions and culture media have not been available [Pomponi S. A., Willoughby R. (1994) In: R. van Soest, A. A. Balkema (Eds.) Sponges in Time and Space, Rotterdam, Brookfield, pp. 395-400; Ilan M., Contini H., Carmeli S., Rinkevich B. (1996) J. Mar. Biotechnol. 4, 145-149]. The media used were supplemented with foetal calf/bovine serum [Pomponi S. A., Willoughby R. (1994) In: R. van Soest, A. A. Balkema (Eds.) Sponges in Time and Space, Rotterdam, Brookfield, pp. 395-400; Ilan M., Contini H., Carmeli S., Rinkevich B. (1996) J. Mar. Biotechnol. 4, 145-149]. So far, it has been assumed that growth factors found in the serum of vertebrates also stimulate the cell growth of sponges and other invertebrates. This assumption does not, however, appear appropriate; the reason for this is our findings which show that sponge cells possess receptors on their surface that are activated by ligands which are different from those of mammalian receptors in general and human receptors in particular. Accordingly, it cannot be regarded as very probable that growth factors which occur in calf/bovine sera will have an effect on sponge receptors specifically and invertebrates in general. Furthermore, media rich in sera entail the risk of contamination with protozoa [Osinga R., Tramper J., Wijffels R. H. (1998) Trends Biotechnol. 16, 130-134].
What is surprising and novel is our finding, which is the subject matter of the present patent application, that in vitro conditions can be defined which lead to the formation of multicellular aggregates of sponges, corals and other invertebrates from dissociated single cells. The aggregates have an appearance similar to tissue and can be maintained in a culture for more than five months. These aggregates are referred to as primmorphs. In addition, we describe how cells, having associated themselves into primmorphs, become telomerase-positive and are capable of DNA synthesis/cell proliferation.